This invention relates to an improved process for the preparation of stable yeast crystals and a process for the enhanced production of ethanol using the said stable yeast crystals. This invention, particularly, relates to a process for the preparation of stable yeast crystals and production of ethanol using these crystals, which has wide industrial application as solvent, food, medicine and intermediate for synthesis of variety of organic compounds.
The demand for ethanol is ever increasing mainly due to the rapid industrialization and fast population growth, which resulted in renewed interest in the development of suitable fermentation processes for ethanol production. Even today, ethanol production is carried out by conventional batch fermentation techniques using Sachhromyces cereviceae cultures. This method of ethanol fermentation with yeast cultures requires maintenance of yeast cultures and pitching of yeast for every batch. Hitherto several attempts have been made to enhance the ethanol production with free and immobilized yeast cells. The ethanol fermentations are generally carried out using Sacchromyces cereviceae in diluted molasses solutions in a batch reactor for a period of 24-48 hours [S. V. Ramakrishna, V. P. Sreedharan and P. Prema. In Bioreactor Immobilized Enzymes and Cells: Fundamentals and Applications (Ed MoodYoung), Elsevier Appl. Sci., Amsterdam, 1988, 251-260]. The major disadvantages of the batch process are its low productivity, extremely slow and inefficient, higher operating as well as capital investment [D. Das. R. G. Nandkishor, K. Murali and P. S. Gupta J. Ferment.Bioengg. 1993, 75, 132-137: D. Weuster-Botz Appl. Microbiol. Biotechnol, 1993, 39, 679-684]. Many alternative fermentation strategies such as high cell densities in the fermentor, larger throughput by continuous mode of operation using cell recycle [C. W. Lee and H. N. Chang Biotehcnol. Bioengg, 1987, 29, 1105-1112], extractive fermentation [M. Minier and G. Goma Biotehcnol. Bioengg. 1982, 24, 1565-1579] and whole cell immobilization by various techniques [S. V. Ramakrishna and R. S. Prakasham Current Science, 1999, 77, 87-100] have been attempted to enhance volumetric productivity of the system. But these methodologies suffer from the drawback that the yeast cells has to be added in every batch. Continuous fermentation with cell recycles [T. K. Ghosh and R. D. Tyagi Biotechnol. Bioengg, 1979, 21; 1387; G. H. Gil, W. J. Jones and T. G. Tomabene Enzyme Microb. Technol., 1991, 13; 390] and vacuume fermentation [G. R. Cysewski and C. R. Wilke Biotechnol, Bioengg, 1977, 19; 1125] have resulted in considerable increase in the productivity. However, the cell recycling system involves considerable cost input for separation of yeast cells from the fermented broth. One of the attractive alternate method received wide attention is the high cell density fermentations. In this regard the reuse of immobilized yeast cells, instead of free cells, has been attempted by several researchers [R. Jamuna and S. V. Ramakrishna Biomass Bioenergy, 1992, 3; 117-119]. Many methods of cell immobilization such as absorption on solid matrices, cross-linking covalent bonding and entrapment have been tried [S. V. Ramakrishna and R. S. Prakasham Curr. Sci., 1999, 77; 87-100] using natural polymers such as agar, agarose, alginates and carageenan and synthetic polymers like polyacrylamide, poly vinyl alcohol have been used for entrapment [S. V. Ramakrishna and R. S. Prakasham Curr. Sci., 1999, 77; 87-100]. Due to toxicity problems, the synthetic polymers have been found to have limited use. One of the major limitation with entrapped cells in natural polymers is its low mechanical strength and its density due to which the entrapped particles tends to float, causing serious engineering problems both in packed and fluidized bed reactor. Recently, T. E. Abraham et all (1990) have developed high density gel beads by incorporating dense inert compounds [T. E. Abraham, J. Rajagopalan, S. V. Ramakrishna and A. D. Damadaran, Indian Patent, 761/DEL/90]. However, the resultant gel beads with improved density were found to have lower mechanical strength due to weak ionic bonds of the polymeric network. There is not report available production of stable immobilized yeast beads. Various types of reactor configurations have been attempted to produce ethanol with entrapped cells [F. Godia, C. Casad, and C. Sola Process Biochem, 1987, 43-48]. One of the major limitation with entrapped cells is that the gas generated during fermentation get entrapped in the gel particles which in turn lowers the density disintegrates the beads [S. V. Ramakrishna, V. P. Sreedharan and P. Prema. In: Bioreactor Immobilized Enzymes and Cells: Fundamentals and Applications (Ed MoodYoung), Elsevier Appl. Sci., Amsterdam, 1988, 251-260].
The first objective of the present invention is to provide an improved process for the preparation of stable yeast crystals.
The second objective of the present invention is the use of the stable yeast crystals prepared by the process of present invention for enhanced production of ethanol.
The third objective of the present invention is to store the stable yeast crystals at room temperature without loosing its activity.
The fourth objective of the present invention is to provide an easy transportation of stable yeast crystals.
The fifth objective of the present invention is to provide stable yeast crystals for repeated use.
The sixth objective of this invention is to provide cost effective improved alcohol fermentation.
The seventh objective of the present invention is to reduce the product inhibition during ethanol fermentation.
The eighth objective of the present invention is to provide an easy process of ethanol production.
The ninth objective of the present invention is to enhance the rate of ethanol fermentation.
The novelty lies in the present invention is the preparation of novel stable yeast crystals which are mechanically strong and biologically active, they can be employed either in stirred tank reactors or in fluidized beds in batch or continuous fermentations, for enhanced ethanol production. The gas generation during fermentation does not lower its density nor weaken the integrity of the matrix.
Accordingly, the present invention provides an improved process for the preparation of stable yeast crystals for enhanced production of ethanol which comprises culturing of yeast (Sacchromyces cereviceae) in a conventional growth medium, separating the yeast, immobilization of yeast using aqueous natural polymer solution by known methods to obtain immobilized yeast beads, separation of the said immobilized yeast beads and dehydration at the temperature in the range of 24-36xc2x0 C. for a period of 2-20 hours to obtain stable yeast crystals having the moisture content in the range between 5-30%, adding novel stable yeast crystals to 5-8% molasses solution and incubating the said crystals for a period ranging between 6-48 hours at a temperature ranging between 24-32xc2x0 C. to obtain activated stable yeast crystals, separating activated stable yeast crystals by conventional methods followed by incubating novel activated stable yeast crystals in the range of 0.2 to 5% of the volume of the fermentation broth containing molasses having a total reducing sugar concentration in the range of 10-30% and recovering the ethanol from the fermentation broth by known methods.
In an embodiment of the present invention, the growth media used comprises (g/1) malt extract, 1.0-5.0; yeast extract, 1.0-5.0; Peptone, 3.0-10.0 and Molasses, 30.0-50.0.
In an other embodiment of the present invention the yeast used is commercially available Sacchromyces species.
In another embodiment of the present invention, the yeast is cultured by incubating at the temperature of 24-36xc2x0 C.
In still other embodiment of the present invention, the pH of the growth medium was maintained in the range of 6.0-7.5 during the growth period.
In still another embodiment of the present invention, the yeast is separated by using conventional methods such as centrifugation, settling, decanting the supernatant, etc. from the growth media.
In yet other embodiment of the present invention, the yeast is immobilized using natural polymers selected from sodium alginate, agar-agar, carageenan etc.
In yet another embodiment of the present invention, the immobilized beads are separated from the solution by decanting the salt solution.
In still yet other embodiment of the present invention the immobilized beads are dehydrated by incubating at a preferred dehydrating temperature 24-36xc2x0 C.
In yet still another embodiment of the present invention, the dehydration is performed for a period ranging between 2-20 hours.
In further embodiment of the present invention, the stable yeast crystals dehydrated until the moisture content in the beads was in the range of 5-30%.
In another embodiment of the present invention, molasses solution having sugar concentration in the range of 3-5% was prepared.
In other embodiment of present invention, the incubation of novel stable yeast crystals is done for a period of 6-48 hours at 24-32xc2x0 C.
In still another embodiment of the present invention, the activated stable yeast crystals were separated from the activation media by draining out the solution or straining through a mesh or perforated bottoms.
In yet another embodiment of the present invention, the activated stable yeast crystals (0.5-2.0%) in respect of the volume of media were added in conventional manner to fermentation broth which was prepared by diluting the molasses with water to adjust the level of fermentable sugar in the range of 15-30% in the final fermentation broth.
In still other embodiment of the present invention, the reaction was carried out with fermentation media for 18-30 hours at 28xc2x12xc2x0 C.
In yet other embodiment of the present invention, the activated clustered yeast crystals were separated after fermentation from the fermentation broth by draining out the fermentation broth.
In yet another embodiment of the present invention, the ethanol recovered from the fermented broth is in the range of 7-15%.
According to the process of the invention, the yeast, Sacchromyces cereviseae, was grown by inoculation in a growth media consisting of (g/1) malt extract-3.0; yeast extract-3.0; Peptone-5.0 and Molasses-30-50. The media was sterilized at 121xc2x0 C. for 15 minutes after pH was adjusted to 6.8-7.2 using 1 normal sodium chloride or 1 normal hydrochloric acid. This was then incubated at 28xc2x12xc2x0 C. on a rotary shaker adjusted with 150 rpm for 24 hours. Large-scale production of yeast was carried out in a fermentor for 24-36 hours with aeration. The yeast was separated by centrifuging the culture broth at 5,000-15,000 rpm for 10 minutes at 24-32xc2x0 C. The yeast slurry was prepared by mixing the yeast 0.5-10% (w/v) with 0.5-3% (w/v) of natural polymer solution and the immobilized yeast beads were prepared by dropping drop by drop with the help of peristatic pump into the curing (0.05-0.3 M CaCl2) solution. These beads were then cured for overnight at 4xc2x0 C. in curing solution. The immobilized yeast beads were then separated by decanting the solution and washed with distilled water thoroughly for 2-3 time. After draining the water, the yeast beads were dehydrated at the temperature 24-36xc2x0 C. for a period of 2-20 hours to obtain stable yeast crystals having a moisture content of 5-30% to obtain stable yeast crystals.
The characteristics of the stable yeast crystals are granular, spherical particles having the diameter 0.3-1.0 mm, dark brown to blackish in colour, hard and robush particles which are insoluble in aqueous or organic medium. These crystals have intrinsic capacity to adsorb and desorb water molecules. These stable yeast crystals were activated by incubating these crystals in the 5-8% molasses solution, consisting of total reducing sugar concentration in the range of 3-5%, at pH 5.0-7.5, for 2-48 hours at temperature 24-32xc2x0 C. The activated stable yeast crystals were then separated from the solution by draining out the aqueous medium. The fermentation broth was prepared by diluting the molasses with water so that the final solution should contain the fermentable sugars in the range of 10-30% and the pH of the fermentation broth was adjusted to 4.0-4.5. Then 0.5-5.0% activated stable yeast crystals were added to the fermentation broth and incubated for 9-48 hours at 24-40xc2x0 C. After fermentation the fermented broth separated by decanting and the ethanol was recovered by known methods.
The present invention is explained with the help of the following examples which are illustrative in nature and should therefore be not construed to limit the scope of the present invention.